The present invention is based, in part, on the discovery that transgenic avians may be produced by in vivo transfection. In particular, the present invention provides methods for producing transgenic avians by delivering nucleic acid (e.g., heterologous nucleic acid) to tissue, such as the oviduct tissue, of a live avian. The present invention provides for, among other things, producing transgenic avians by in vivo electroporation of avian tissue and by applying nucleic acid to avian tissue under pressure.
Transgenic technology to convert animals into “bioreactors” for the production of specific proteins or other substances of pharmaceutical interest (Gordon et al., 1987, Biotechnology 5: 1183-1187; Wilmut et al, 1990, Theriogenology 33: 113-123) offers significant advantages over more conventional methods of protein production by gene expression. Recombinant nucleic acid molecules have been engineered and incorporated into transgenic animals so that an expressed heterologous protein may be joined to a protein or peptide that allows secretion of the transgenic expression product into milk or urine, from which the protein may then be recovered. These procedures, however, may require lactating animals, with the attendant costs of maintaining individual animals or herds of large species, such as cows, sheep, or goats.
Historically, transgenic animals have been produced mostly by microinjection of the fertilized egg. The pronuclei of fertilized eggs are microinjected in vitro with heterologous DNA or hybrid DNA molecules. The microinjected fertilized eggs are then transferred to the genital tract of a pseudopregnant female (e.g., Krimpenfort et al., U.S. Pat. No. 5,175,384).
One system that holds potential is the avian reproductive system. The production of an avian egg begins with formation of a large yolk in the ovary of the hen. The unfertilized oocyte or ovum is positioned on top of the yolk sac. After ovulation, the ovum passes into the infundibulum of the oviduct where it is fertilized, if sperm are present, and then moves into the magnum of the oviduct which is lined with tubular gland cells. These cells secrete the egg-white proteins, including ovalbumin, lysozyme, ovomucoid, conalbumin and ovomucin, into the lumen of the magnum where they are deposited onto the avian embryo and yolk. The hen oviduct offers outstanding potential as a protein bioreactor because of the high levels of protein production, the promise of proper folding and post-translation modification of the target protein, the ease of product recovery, and the shorter developmental period of chickens compared to other potential animal species.
Various techniques have been used in efforts to create transgenic chickens expressing heterologous proteins in the oviduct.
Bosselman et al. in U.S. Pat. No. 5,162,215 describes a method for introducing a replication-defective retroviral vector into a pluripotent stem cell of an unincubated chick embryo, and further describes chickens whose cells express a heterologous vector nucleic acid sequence. Such retroviral vectors have significant limitations, for example, only relatively small fragments of nucleic acid can be inserted into the vectors precluding, in most instances, the use of large portions of the regulatory regions or introns of a genomic locus which can be useful in obtaining significant levels of heterologous protein expression. Additionally, retroviral vectors may be inappropriate for generating transgenics for the production of pharmaceuticals due to safety and regulatory issues.
In addition, previously described methods of producing transgenic avians may not always allow for the targeting of nucleic acid of interest to the desired tissue in the avian, for example, the oviduct tissue. Further, many techniques currently available for producing a transgenic chicken require the time necessary for a chicken to develop from a blastoderm to egg laying maturity.
What is needed are methods of introducing large segments of nucleic acid into avian cells and methods for the targeting nucleic acid to specific tissue in the avian for example, the oviduct tissue. Further, what is needed are methods of producing transgenic avians for which a prolonged time necessary to achieve egg-laying maturity is not required.